Jonathan A Cooper
Fred Hutchinson Cancer Research Center
Project start date: 1986-01-01
Project end date: 2017-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Jonathan A Cooper
Src Family Kinases And Cell Growth Regulation
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 2R01CA041072-22 from National Cancer Institute IRG: NCF
Abstract: We propose to continue our studies on the tyrosine kinase Src (also called c-Src), the cellular homolog of the v-Src oncoprotein. Activated Src causes transformation of some cell types and cancer in animals, but can stimulate differentiation, migration, survival or apoptosis when expressed in other cell types. Because activated Src phosphorylates many different cell proteins, the specific mechanisms underlying these different cellular outcomes have been difficult to dissect. Moreover, the roles of the normal cellular Src protein have been unclear, in part due to overlapping functions between Src and other close relatives. Src activity is elevated in many human cancers, including approximately 70% of carcinomas. Understanding a novel aspect of Src biology is the general goal of this proposal. Over the past funding period, our study of a Src substrate, Dab1, led us to uncover a Src-dependent signaling pathway that regulates the migrations of neuronal precursors in the developing mammalian central nervous system. An extracellular protein, Reelin, acts via non-catalytic receptors to stimulate Src- dependent tyrosine phosphorylation of Dab1. Genetic evidence shows that tyrosine phosphorylation of Dab1 is both a cause and a consequence of activation of Src, and both events are required for downstream signaling. Reelin stimulates Dab1 binding to several identified signaling proteins, including the adaptor proteins Crk and CrkL. Reelin also stimulates Dab1 ubiquitylation and degradation, and Reelin receptor endocytosis. However, it is not yet clear whether Dab1 ubiquitylation regulates signaling or endocytosis or is a feedback control, and it remains unclear how Dab1 degradation, Reelin endocytosis, Dab1-complex formation, and Src kinase activation link to each other and regulate neuron precursor movements in vivo. We propose to use genetic and biochemical approaches to uncover the mechanisms by which Src and Dab1 regulate cell migrations in vivo. We propose two broad aims to uncover the importance of different Dab1 phosphorylation sites and signaling complexes and Dab1 degradation in Dab1 regulation and development; and to determine the specific roles of Crk, CrkL, their downstream mediator C3G, and endocytosis in signaling. By further study of this signaling process, we expect to identify principles of signal complex formation and regulation that may be relevant to other tyrosine kinase signaling pathways.
Keywords: cell growth regulation, endocytosis, family, phosphorylation, protein, tyrosine, ubiquitin, allele, apoptosis, binding protein, biology, brain, carcinoma, cell, cell migration, cell transformation, cell type, central nervous system, culture, extracellular, gene mutation, genetically modified animal, genetics, human, laboratory mouse, lead, ligase, molecular genetics, mutant, neoplasm /cancer, nervous system, neuron, oncoprotein, protein binding, receptor, role, role model, spinal cord
Project start date: 1986-01-01
Project end date: 2011-12-31
2R01CA041072-22 (2007): $537829
RECEPTOR TYROSINE KINASES: BIOLOGY AND CANCER
Jonathan A Cooper, Member
Federation Of Amer Soc For Exper Biology, 9650 Rockville Pike, Bethesda, Md 20814-3998
Grant 1R13CA150454-01 from National Cancer Institute
Abstract: We are seeking partial support for FASEB Summer Research Conferences entitled "Receptor Tyrosine Kinases Biology and Cancer", to be held June 27 - July 2 2010 and again in June 2012. Such a conference is timely because of exciting recent developments in receptor cross talk, therapeutic advances, and deeper understanding of Receptor Tyrosine Kinases (RTKs) in the brain and development. The planned conferences follow a series of five FASEB conferences on RTKs that were held between 1999 and 2007. RTKs are important in many aspects of animal cell biology, including development, neuroscience and cancer. Previous conferences had an emphasis on RTKs of the Met and ErbB families, and the conferences had seen a dwindling attendance over the years. We have taken several steps to revitalize the meetings. First, while retaining an emphasis on RTKs and cancer, we have widened the scope of the meeting to include other RTKs, other signaling mechanisms, new advances in therapeutic interventions and developmental and neuronal systems in which RTKs play a major role. This broader scope is expected to attract participants with more diverse interests than previously. Second, we have rescheduled the meetings to even numbered years. The previous timing generated conflicts with two other conferences, one sponsored by FASEB and one by Cold Spring Harbor, which addressed similar topics to the RTK conference. These changes, combined with improved advertising and outreach, and an exciting slate of speakers, will boost attendance and ensure the success of the new meetings. FASEB Conferences are small (100-150 participants) meetings held in intimate settings that maximize interactions between senior and junior scientists from academia and industry. In addition to invited speakers, we will intersperse the plenary sessions with short talks selected from the s. 22 of the 33 invited speakers have not presented at this conference ever or in the last 5 years. They include a healthy mix of established and newly-independent investigators, male and female, national and international, addressing basic and applied issues in RTK Biology and Cancer. Refreshments will be provided at the poster sessions to encourage full participation. Financial support is requested to offset travel costs for invited speakers and to fund the travel costs and conference fees for 4 graduate student or postdoctoral level conferees. With this support, we anticipate that the conference will succeed in its goal of promoting scientific exchange and stimulating new research in this important area. This grant requests funding to partly support conferences focused on the roles of RTKs in normal biology and cancer. Receptor tyrosine kinases (RTKs) are molecules on the cell surface that receive signals from the outside and transmit them to the inside of the cell. They share an enzymatic activity that allows them to phosphorylate cell proteins on tyrosine when their extracellular regions are stimulated. There are many different RTKs in animal cells, and the particular RTKs that are expressed by a cell are important for the normal functions of that cell and the organization of tissues during development. In the nervous system, RTKs respond to signals that guide neuronal connections and ensure their survival. During embryonic development, RTKs help coordinate tissue and organ development by regulating cell proliferation, migration and differentiation. RTKs are frequently abnormal in cancer cells. RTKs are also important drug targets for cancer chemotherapy. The conferences will be open to scientists at all stages of their careers, and funding is sought to offset the travel costs of invited speakers and also of 4 student or postdoctoral participants
Keywords: ATP[{..}]protein-tyrosine O-phosphotransferase; Academia; Address; Advertising; Animals; Area; Body Tissues; Brain; Cancer Biology; Cancers; Cell Communication and Signaling; Cell Function; Cell Growth in Number; Cell Multiplication; Cell Process; Cell Proliferation; Cell Signaling; Cell physiology; Cell surface; Cells; Cellular Function; Cellular Physiology; Cellular Process; Cellular Proliferation; Cellular biology; Chemotherapy Protocol; Chemotherapy Regimen; Chemotherapy, Cancer, General; Chemotherapy-Oncologic Procedure; Combination Chemotherapy Regimen; Conflict; Conflict (Psychology); Development; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drug Targetings; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Embryo Development; Embryogenesis; Embryonic Development; Encephalon; Encephalons; Ensure; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Family; Fees; Female; Financial Support; Funding; Goals; Grant; HEK3; Industry; International; Intracellular Communication and Signaling; Investigators; L-Tyrosine; Malignant Cell; Malignant Neoplasms; Malignant Tumor; NRVS-SYS; Nerve Cells; Nerve Unit; Nervous System; Nervous System, Brain; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurons; Neurosciences; Organ; PTK; PTK Receptors; Participant; Play; Posters; Posters [Publication Type]; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Proteins; Quimioterapia; RTK; Receptor Cross-Talk; Receptor Protein; Receptor Protein-Tyrosine Kinases; Research; Research Personnel; Researchers; Role; Scientist; Series; Signal Transduction; Signal Transduction Systems; Signaling; Staging; Students; Subcellular Process; System; System, LOINC Axis 4; TYR; Therapeutic; Therapeutic Intervention; Time; Tissues; Transmembrane Receptor Protein Tyrosine Kinase; Travel; Tyrosine; Tyrosine Kinase; Tyrosine Kinase Growth Factor Receptor; Tyrosine Kinase Linked Receptors; Tyrosine Kinase Receptors; Tyrosine, L-isomer; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; ing; biological signal transduction; cancer cell; cancer chemotherapy; career; cell biology; conference; cost; extracellular; gene product; graduate student; hydroxyaryl protein kinase; improved; interest; intervention therapy; male; malignancy; meetings; migration; neoplasm/cancer; neuronal; outreach; para-Tyrosine; posters; public health relevance; receptor; social role; success; symposium; tyrosyl protein kinase
Relevance: This grant requests funding to partly support conferences focussed on the roles of RTKs in normal biology and cancer. Receptor tyrosine kinases (RTKs) are molecules on the cell surface that receive signals from the outside and transmit them to the inside of the cell. They share an enzymatic activity that allows them to phosphorylate cell proteins on tyrosine when their extracellular regions are stimulated. There are many different RTKs in animal cells, and the particular RTKs that are expressed by a cell are important for the normal functions of that cell and the organization of tissues during development. In the nervous system, RTKs respond to signals that guide neuronal connections and ensure their survival. During embryonic development, RTKs help coordinate tissue and organ development by regulating cell proliferation, migration and differentiation. RTKs are frequently abnormal in cancer cells. RTKs are also important drug targets for cancer chemotherapy. The conferences will be open to scientists at all stages of their careers, and funding is sought to offset the travel costs of invited speakers and also of 4 student or postdoctoral participants
Project start date: 2010-06-01
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-08-149
1R13CA150454-01 (2010): $8000
CHROMOSOME METABOLISM AND CANCER
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5T32CA009657-15 from National Cancer Institute IRG: NCI
Abstract: This renewal application is seeking support for a program that trains researchers to understand the links between chromosome metabolism and cancer. Recent advances in the studies of genes and genomes have greatly facilitated the understanding of chromosome metabolism and illustrated its importance in cancer cell formation and progression. The 27 training faculty are experts in this area from the Basic Sciences Division and the Human Biology Division of Fred Hutchinson Cancer Research Center. Many are leading scientists in their own fields, and together they form a cohesive group that approaches the issue from different but complementing angles. They include structural biologists in addition to molecular and cellular biologists, and utilize various model organisms such as nematode, fly, yeast in addition to vertebrates and cultured cells in their studies. The breadth and depth of this program should help generate excellent young scientists ready to deal with this complex problem. The program will continue to train at the predoctoral and postdoctoral levels. Three predoctoral positions are requested to maintain the current level of support of this successful program that was initiated five years ago following the formation of the Molecular and Cell Biology (MCB) Graduate Program. The postdoctoral positions will be expanded to six (from the current four positions) due to increased interests in this area and the expanded training faculty of the program (from 14 to 27). Predoctoral trainees will be recruited among the MCB students, who enter the joint program with the University of Washington, through vigorous competition and are among the best in the nation. Postdoctoral trainees will be recruited nationally. Training of predoctoral students includes course work, laboratory rotations and thesis research. Postdoctoral training will focus on independent research. All t r ainees will benefit from the rich activities available for cancer researchers at this Center. They will participate in weekly research group meetings, joint group meetings and Division meetings to report research findings to and exchange ideas with scientists within the lab, labs with similar interests, or the entire Divisions. They will also benefit from the strong interdisciplinary programs in the Center that expose basic scientists to clinical and public health aspects of cancer. In addition, they will have ready access to the newly formed patient care unit on campus. It is hoped that this program will train young scientists with in-depth understanding of chromosome metabolism and able to explore its various links to cancer cause, care and prevention.
Project start date: 1991-04-01
Project end date: 2006-03-31
5T32CA009657-15 (2005): $343370
5T32CA009657-14 (2004): $330752
3T32CA009657-19S1 (2009): $214356
CHROMOSOME METABOLISM AND CANCER TRAINING GRANT
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center, Po Box 19024, Seattle, Wa 98109-1024
Grant 5T32CA009657-20 from National Cancer Institute
Abstract: This application seeks continuing support for the on-going Chromosome Metabolism and Cancer Training Program that trains researchers exploring the links between chromosome metabolism andcancer. Advances in the studies of genes and genomes have greatly facilitated the understanding of chromosome metabolism (gene expression, chromatin assembly, segregation, telomeres and centromeres, DNA repair, etc.) and illustrated its importance in cancer initiation and progression. The 25 training faculty are experts in this area from the Basic Sciences Human Biology Divisions of the Fred Hutchinson Cancer Research Center. Many are leading scientists in their own fields, and together they form a cohesive group that approaches the issue from different but complementing angles. They include structural biologists in addition to molecular and cellular biologists, and utilize various model organisms such as nematode, fly and yeast in addition to vertebrates and cultured cells in their studies. The breadth and depth of this program should help generate excellent young scientists with creative approaches to understanding cancer mechanisms. Two predoctoral positions are requested to maintain the current level of support of this successful program that was initiated ten years ago following the formation of the Molecular and Cell Biology (MCB) Graduate Program. The postdoctoral positions will also be maintained at the current level of five. Predoctoral trainees will be recruited among the MCB students, who enter the PhD program through vigorous competition and are among the best in the nation. Postdoctoral trainees are drawn from an outstanding pool of fellows working with CMTP Faculty. Predoctoral students are appointed to the training grant after they have chosen a permanent laboratory and completed lab rotations and a portion of their required coursework. Postdoctoral training consists of independent research, supplemented by rigorous quarterly presentations in front of training grant faculty and experience in organizing CMTP-sponsored seminars. In addition, all trainees will benefit from the rich activities available for researchers at this Center, including the strong interdisciplinary programs that expose basic scientists to clinical and public health aspects of cancer. It is hoped that this program will create young scientists with in-depth understanding of chromosome metabolism and commitment to explore its various links to cancer cause, cure and prevention
Keywords: Cancers; Chromosomes; Grant; Intermediary Metabolism; METBL; Malignant Neoplasms; Malignant Tumor; Metabolic Processes; Metabolism; Training; malignancy; neoplasm/cancer
Project start date: 1991-04-01
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
5T32CA009657-20 (2010): $265390
5T32CA009657-19 (2009): $302010
5T32CA009657-17 (2007): $212494
2T32CA009657-16 (2006): $341180
SRC FAMILY KINASES AND CELL GROWTH REGULATION
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01CA041072-13 from National Cancer Institute IRG: CBY
Abstract: The src gene is a proto-oncogene whose product, the Src protein, is representative of a sub-group of intracellular protein tyrosine kinases (the Src family). Mutations in the src gene that deregulate the tyrosine kinase activity of Src are oncogenic. Src family kinases are negatively regulated by a number of mechanisms, including an inhibitory C-terminal tyrosine phosphorylation. Normally, the stoichiometry of this phosphorylation is high because the rate of phosphate transfer to Src exceeds the rate of dephosphorylation. Clearly, it is important to identify and analyze the regulation of the kinases and phosphatases involved in these reactions in order to understand how Src family kinases are regulated. We have been characterizing a kinase, Csk, that specifically phosphorylates Src at the inhibitory site. We have now developed antibodies, in vitro kinase assays, and DNA constructs for expressing Csk. We have found that the intracellular localization of Csk is regulated by Src. One goal of this proposal is to study the mechanism of this localization, and to determine the significance of Csk translocation for its ability to repress Src. Another goal is to determine the functions of individual domains within Csk for regulation of Src. We will search for homologs of Csk. Finally, we are interested in the role of the Src homology 3 (SH3) domain of Src in Src regulation and transformation. We are screening for cell proteins that interact with the Src SH3 domain and propose experiments to analyze these interactions.
Keywords: cell growth regulation, enzyme induction /repression, oncogene, phosphorylation, protein structure /function, protein tyrosine kinase, binding protein, enzyme activity, gene expression, gene mutation, genetic regulatory element, phosphoprotein, SDS polyacrylamide gel electrophoresis, immunoprecipitation, laboratory rabbit, nucleic acid hybridization, nucleic acid probe, nucleic acid sequence, polymerase chain reaction, tissue /cell culture, western blotting
Project start date: 1986-01-01
Project end date: 1998-12-31
5R01CA041072-13 (1998): $292464
Sponsored Links Excellgen http://Excellgen.com
5R01CA041072-12 (1997): $281298
5R01CA041072-10 (1995): $246885
5R37CA041072-16 (2001): $434308
5R37CA041072-15 (2000): $434308
2R37CA041072-14 (1999): $395516
SIGNAL TRANSDUCTION VIA RECEPTOR TYROSINE KINASES
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01CA054786-04 from National Cancer Institute IRG: CBY
Abstract: The overall goal is to elucidate mechanisms of signal transduction through growth factor receptors. Previous results showed that stimulation of the platelet derived growth factor (PDGF) receptor with PDGF triggers the physical binding of a number of cell proteins to the receptor. Two of these proteins are phosphatidylinositol (PI) 3 kinase and the Ras GTPase activator protein (GAP). Mapping of tyrosine phosphorylation sites in the PDGF receptor, mutagenesis, and in vitro biochemical studies have provided evidence that binding of GAP and PI3 kinase involves receptor phosphorylation, activated by PDGF. These results lead to the hypothesis that binding and phosphorylation of PI3 kinase, GAP and the other cell proteins by the PDGF receptor is involved in initiating signalling events within the cells, culminating in a mitogenic response. The work will be extended in three directions (1) Investigation of other sequences in the PDGF receptor, besides the identified phosphorylation sites, that are important for binding reactions. The results should contribute to the design of molecules that may inhibit the binding and phosphorylation reactions, and may inhibit signal transduction through the PDGF receptor. (2) Test whether results obtained with the PDGF receptor extend to another receptor tyrosine kinase, Kit, a protein of general interest because of its role in hematopoiesis. (3) Perform experiments with GAP to try to understand how its binding to the receptor occurs and how binding and phosphorylation of GAP may be involved in signal transduction.
Keywords: biological signal transduction, growth factor receptor, platelet derived growth factor, protein tyrosine kinase, receptor binding, chemical binding, chimeric protein, guanine nucleotide binding protein, guanosinetriphosphatase, mitogen, oncogene, phosphatidylinositol, phosphorylation, protein structure, animal tissue, immunoprecipitation, laboratory rabbit, radionuclide, site directed mutagenesis, tissue /cell culture, western blotting
Project start date: 1991-09-01
Project end date: 1995-08-31
5R01CA054786-04 (1994): $146149
5R01CA054786-03 (1993): $139878
5R01CA054786-02 (1992): $135397
In Vivo Functions Of Possible Tumor Suppressor Dab2
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01GM066257-04 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: The Disabled-2 (Dab2) gene inhibits tumor growth through unknown mechanisms. There are 2 Dab2 protein forms, p96 and p67 that appear to be adaptor proteins. Some adaptor proteins regulate endocytosis and others function in signal transduction. One exciting idea that we ll test for Dab2 is that some proteins do both. Our overall goal is to define the molecular mechanisms underlying the in vivo functions of Dab2. We ve found that p96 but not p67 binds to endocytic proteins and localizes to clathrin-coated pits. In addition, both p96 and p67 contain a PTB/PID domain that associates with the endocytosis signals of lipoprotein receptors, and a separate domain that binds to a non-muscle myosin, myosin VI, that has been implicated in endocytosis. Indeed, we ve shown that lack of Dab2 in the kidney causes protein transport defects similar to those of mice lacking the lipoprotein receptor, megalin. Thus we hypothesize that Dab2 normally regulates megalin traffic. In addition, we ve found that Dab2 has another important function prior to gastrulating, when p67 is the predominant form. Dab2 mutant embryos have a defect in an extraembryonic epithelium, the visceral endoderm, and this defect prevents induction of the anterior posterior axis. The phenotype appears to result from impaired signaling by Nodal, a TGFbeta family member. We hypothesize that p67 is involved in signaling in the visceral endoderm. Since p67 lacks signals for coated pit localization, it is possible it is directly involved in signal transduction. We will identify the defective signaling pathways in Dab2-mutant visceral endoderm, and identify the step requiring Dab2. We will test whether p96 is specialized to regulate kidney transport and p67 is specialized for signal transduction, or whether each form is multifunctional. Coupled with studies on subcellular localization of Dab2 proteins in kidney and visceral endoderm, this approach will provide evidence whether the embryonic requirement for Dab2 is an indirect consequence of a role in receptor trafficking, or whether p67 has a signaling function. We will investigate the mechanism by which Dab2 regulates megalin traffic in the kidney, especially the importance of binding to components of clathrin-coated pits and myosin VI. We ve also found that Dab2 is phosphorylated in mitogen-stimulated cells by MAP kinase, and will investigate the significance of Dab2 phosphorylation in regulating biological responses.
Keywords: biological signal transduction, embryogenesis, endoderm, protein structure function, protein transport, tumor suppressor gene, endocytosis, kidney cell, mitogen activated protein kinase, myosin, phosphorylation, renal tubule, SDS polyacrylamide gel electrophoresis, gene targeting, genetically modified animal, immunocytochemistry, laboratory mouse, polymerase chain reaction, southern blotting, tissue /cell culture, western blotting
Project start date: 2003-06-01
Project end date: 2008-05-31
5R01GM066257-04 (2006): $371656
5R01GM066257-03 (2005): $478440
Sponsored Links Excellgen http://Excellgen.com
5R01GM066257-02 (2004): $475591
1R01GM066257-01A1 (2003): $380600
3R01GM066257-01A1S1 (2004): $10923
PROTEIN PHOSPHORYLATION AND CELL GROWTH REGULATION
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01CA041072-03 from National Cancer Institute IRG: CBY
Abstract: The aim of this investigation is to understand how the phosphorylation of tyrosine hydroxyls in cellular proteins may be involved in the regulation of vertebrate cell growth in culture. Previous experiments by the Principal Investigator and others have established that various mitogens measurable increase the proportion of phosphate that is esterified to proteins via tyrosine. A group of specific cellular proteins, of unknown function, are phosphoylated de novo at tyrosine in response to many mitogens. Some mitogens bind to cell surface receptors that posses tyrosine protien kinase activity, so the proteins we identified may interact directly with these mitogen receptors. Other mitogens whose receptors appear not to be tyrosine protein kinases induce protein phosphorylation by unknown mechanisms. Tyrosine protein kinase activity is also a function of the transforming proteins of some retroviruses, and may be important for the unrestrained proliferation of the cells they transform. It seems likely that since phosphorylation of cell proteins at tyrosine correlates with mitogenesis it is one of several events necessary to trigger proliferation of resting cells. In the first project, a major substrate for mitogen-activated tyrosine protein kinases will be purified, characterized, and used for partial sequence analysis and to immunize animals. A cDNA clone for the protein will be obtained and its sequence derived. The function of the protein in the cell, and its interactions with other cell proteins that may be involved in relay of the mitogenic signal to the nucleus, will be probed using the twin tools of specific antibodies and expression of recombinant DNA clones. In a second project, we will characterize the tyrosine protein kinase(s) that is activated by two classes of mitogens, tumor promoters and mitogenic proteases, whose receptors are not known to possess tyrosine protein kinase activity. This tyrosine protein kinase may be activated following phosphorylation by protien kinase C, the major receptor for tumor promoters in the cell.
Keywords: CELL DIVISION, MITOSIS, MITOGENS, ENZYME INDUCTION-REPRESSION-DEREPRESSION, NEOPLASTIC TRANSFORMATION, CARCINOGENS, TUMOR PROMOTERS, PHOSPHOTRANSFERASES-ATP, PROTEIN KINASES, PROTEIN-TYROSINE-KINASE, cell growth regulation, phosphorylation, ENZYME MECHANISMS, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, ONCOGENES, GENETICS, MUTATION, GENE MUTATION, GROWTH FACTORS (INCL. ANABOLICS), PLATELET DERIVED GROWTH FACTOR, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, PROTEIN (PEPTIDE) SEQUENCE, PROTEINS, PHOSPHOPROTEINS, RECEPTORS, GROWTH FACTOR RECEPTORS, protein metabolism, CHEMICAL BONDS, CROSSLINKS, DOSAGE AND ROUTE, INJECTIONS, MICROINJECTIONS, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, PRECIPITIN TECHNIQUE, IMMUNOLOGY, ANTIBODIES, IMMUNE SERA, NUCLEIC ACIDS, COMPLEMENTARY DNA, NUCLEIC ACIDS, NUCLEIC ACID PROBES, PHYSICAL SEPARATION, CHROMATOGRAPHY, AFFINITY, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, RADIOAUTOGRAPHY, TISSUE (CELL) CULTURE
Project start date: 1986-01-01
Project end date: 1988-12-31
5R01CA041072-08 (1993): $178846
5R01CA041072-07 (1992): $169399
SIGNAL TRANSDUCTION VIA RECEPTOR TYROSINE KINASES
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center Box 19024, 1100 Fairview Ave N Seattle, Wa 981091024
Grant 5R01CA054786-09 from National Cancer Institute IRG: CBY
Abstract: Our broad objective is to trace the pathways utilized by receptor tyrosine kinases to regulate cell proliferation and to understand how these pathways are altered in malignancy. Previous studies, from this and other laboratories, have shown that specific cell proteins bind to phosphorylated tyrosine residues in activated receptor tyrosine kinases. The bound proteins regulate different signaling pathways. One pathway leads to the activation of the membrane-associated GTPase, Ras, by promoting the release of GDP from Ras and its replacement by GTP. In its GTP state, Ras binds protein kinases of the Raf family. The membrane-associated Ras-Raf complex is a substrate for one or more other regulatory inputs that together stimulate Raf kinase activity. One known substrate of Raf is responsible for MAP kinase activation, but Raf may have other substrates. We have identified three other proteins capable of specific interactions with RasGTP. These proteins are a Ral guanine nucleotide dissociation stimulator (GDS), a protein related to the RalGDS, and a protein related to the catalytic (P110) subunits of phosphoinositide 3-kinases. We propose to confirm that these proteins interact with Ras GTP in the cell, determine the biochemical functions of these proteins, and evaluate their roles in oncogenic transformation by activated mutants of Ras. Another goal of this proposal is to identify residues on Ras and Raf involved in binding and generate tools for selective activation of the Raf pathway and other Ras-dependent pathways.
Keywords: biological signal transduction, cell growth regulation, gene expression, neoplastic transformation, oncogene, protein structure /function, protein tyrosine kinase, enzyme activity, guanine nucleotide binding protein, guanosinetriphosphatase, mitogen activated protein kinase, phosphatidylinositol, phosphorylation, receptor binding, antisense nucleic acid, immunoprecipitation, laboratory rabbit, molecular cloning, northern blotting, nucleic acid sequence, site directed mutagenesis, tissue /cell culture, western blotting
Project start date: 1995-09-26
Project end date: 2001-06-30
5R01CA054786-09 (1999): $274502
Sponsored Links Excellgen http://Excellgen.com
5R01CA054786-08 (1998): $287145
5R01CA054786-07 (1997): $272971
2R01CA054786-05 (1995): $242343
INTEGRATION OF STRESS AND MITOGEN SIGNALING
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center
box 19024, 1100 Fairview Ave N
seattle, Wa 981091024
Grant 5R01CA073897-05 from National Cancer Institute IRG: CBY
Abstract: Numerous studies have demonstrated that mitogen-activated protein kinase (MAPK) cascades link environmental signals to various cellular responses. Recent work by has focused on searching for MAP kinase substrates and has led to the identification of a potentially new class of protein-serine/threonine kinases that are associated with, and apparently activated by, specific MAP kinases. This class includes at least two closely related molecules, termed Msk1 and Msk2, and at least one other more distant member that also appears to be a kinase, i.e., Mapkip 108. Based on preliminary studies, proposes a model of Msk regulation wherein inactive Msk exists in a complex with inactive ERK (mitogen-regulated) and p38 (stress-activated) MAP kinases in the cell. In response to certain extracellular signals, the MAP kinases (ERK and p38, but not JNK) phosphorylates and activates the bound Msk. Thus, the overall goal of these studies is to assess the significance of these new MAP kinase substrates in regulating cell proliferation and stress responses. To this end, the following five Specific Aims are proposed 1) To establish the mechanism of Msk activation by developing a Msk assay and ascertaining how ERK and p38 activation influences Msk (principally Msk2) phosphorylation and kinase activity. 2) To investigate the relevance of Msk regulation by ERK or p38 for the cellular response to mitogens, oncogenes, developmental cues, or stresses. These studies will use over-expressed or mutated Msk2 and analyses of cell behavior such as foci formation, actin reorganization and sensitivity to oncogene transformation. 3) To identify the molecular targets of Msk via an assessment of the subcellular localization of Msk together with approaches such as the use of a peptide library for kinase specificity determinations and the yeast two hybrid system for examining protein-protein interactions. 4) To map the interactions between ERK and Msk using approaches such at the two-hybrid system. 5) To clone Mapkip108 using conventional techniques
Keywords: biological signal transduction, cell proliferation, enzyme activity, mitogen, mitogen activated protein kinase, stress chemical binding, enzyme substrate, growth factor, oncogene, phosphorylation, protein protein interaction 3T3 cell, SDS polyacrylamide gel electrophoresis, gel mobility shift assay, immunofluorescence technique, immunoprecipitation, laboratory rabbit, molecular cloning, transfection, western blotting
Project start date: 1997-07-01
Project end date: 2002-04-30
5R01CA073897-05 (2001): $241122
5R01CA073897-04 (2000): $234100
5R01CA073897-03 (1999): $227280
1R01CA073897-01 (1997): $215568
FUNCTIONS AND MECHANISMS OF THE ENDOCYTIC ADAPTOR DAB2
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center, Po Box 19024, Seattle, Wa 98109-1024
Grant 5R01GM066257-07 from National Institute Of General Medical Sciences
Abstract: Endocytosis brings essential nutrients and other molecules into cells, regulates signaling by cell-surface receptors, and modulates interactions with the extracellular environment. Our general goal is to understand how endocytosis affects phenotypes and disease. Disabled 2 (Dab2) is a "phosphotyrosine-binding" (PTB) domain protein that binds to clathrin and other endocytic proteins and localizes to clathrin-coated pits. We have now shown that Dab2 knockout inhibits endocytosis in vivo and in vitro. Dab2 functions as a clathrin- associated sorting protein (CLASP) for low density lipoprotein receptors (LDLRs). Unlike all previously characterized CLASPs, Dab2 function is independent of AP-2. We also found that Dab2 depletion from normal cells affects adhesion, migration and integrin endocytosis. Dab2 is known to be strongly down-regulated in many different human carcinomas, particularly ovarian and mammary tumors. Loss of Dab2 allows cancer cells to resist anoikis (detachment-induced apoptosis). We would like to understand how Dab2 could cause such phenotypic changes through its role in endocytosis. The proposed research builds on a new technology that allows us to identify receptors trafficked by a given adaptor protein. This approach has revealed that integrin endocytosis requires Dab2. Changes in integrin levels correlate with Dab2-induced changes in focal adhesion turnover during cell spreading and migration. Dab2 is the first adaptor protein found for integrin endocytosis. We are now in a unique position to study the importance of integrin endocytosis for cell adhesion, cytoskeletal organization and motility. We hypothesize that Dab2-dependent cell surface proteins may be up-regulated on the surface when Dab2 is absent, and may be responsible for reduced migration and increased anoikis-resistance of Dab2-deficient cells. We now propose two aims, one global and one specific. In the first Aim, we will identify cargoes requiring different PTB adaptor proteins, Dab2, ARH and Numb, determine how cargoes are selected, and identify internalization signals. We will explore how specific cargoes relate to the cellular phenotypes of Dab2, ARH and Numb, including the effects of Dab2 re-expression in cancer cells. In the second Aim, we will investigate Dab2-mediated endocytosis of integrins. We will ask how integrins are recognized for endocytosis; whether Dab2 mediates basal or adhesion-dependent internalization; whether Dab2-mediated integrin uptake is regulated by growth factors or the cytoskeleton; how integrin endocytosis relates to cell migration, adhesion and cytoskeletal remodeling. We will test whether cancer cells in which Dab2 is down-regulated have increased surface levels of integrins and the role of integrin up-regulation in the cancer phenotype. These studies will enhance our understanding of how receptors and integrins are selected for endocytosis, and the role of cell surface proteins in cell biology and the progression of cancer. Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility. PUBLIC HEALTH REVELANCE Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility
Keywords: AP-2; AP-2 Alpha Gene; AP2 Gene; AP2TF; Activator Protein 2 Gene; Adaptor Protein; Adaptor Signaling Protein; Adhesion Plaques; Adhesions; Affect; Anoikis; Apoptosis; Apoptosis Pathway; Assay; Binding; Binding (Molecular Function); Binding Sites; Bioassay; Biologic Assays; Biological; Biological Assay; Biology; Breast Neoplasms; Breast Tumors; Cancers; Carcinoma; Cell Adhesion; Cell Communication and Signaling; Cell Death, Programmed; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cell Surface Proteins; Cell Surface Receptors; Cell Survival; Cell Viability; Cell surface; Cell-Matrix Adherens Junctions; Cells; Cellular Adhesion; Cellular Matrix; Cellular Migration; Cellular biology; Clathrin; Clathrin Adaptors; Clathrin Assembly Proteins; Clathrin-Associated Adaptors; Clathrin-Associated Proteins; Combining Site; Cytoskeletal Modeling; Cytoskeletal Organization; Cytoskeletal Organization Process; Cytoskeletal Reorganization; Cytoskeletal System; Cytoskeleton; Defect; Development; Disabled Persons; Disabled Population; Endocytosis; Environment; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Extracellular Matrix, Integrins; Focal Adhesions; Focal Contacts; GFAC; Goals; Growth Agents; Growth Factor; Growth Factors, Proteins; Growth Substances; Handicapped; Human; Human, General; Hypercholesteremia; In Vitro; Integrins; Intracellular Communication and Signaling; Knock-out; Knockout; L-tyrosine, dihydrogen phosphate (ester); Lipoprotein LDL Receptors; Low Density Lipoprotein Receptor; Malignant Cell; Malignant Neoplasms; Malignant Tumor; Mammary Cancer; Mammary Neoplasms; Man (Taxonomy); Man, Modern; Maps; Mediating; Membrane Proteins; Membrane-Associated Proteins; Molecular Interaction; Motility; Motility, Cellular; Normal Cell; Nutrient; Ovarian; PTB Domain; People with Disabilities; Persons with Disabilities; Phenotype; Phosphotyrosine; Phosphotyrosine Binding Domain; Position; Positioning Attribute; Protein Binding; Proteins; Public Health; Reactive Site; Receptor Protein; Receptors, LDL; Recycling; Regulation; Research; Resistance; Role; Signal Transduction; Signal Transduction Systems; Signaling; Sorting - Cell Movement; Surface; Surface Proteins; TFAP2; TFAP2A; TFAP2A gene; Testing; Tumor Suppressor Proteins; Tyrosine-O-phosphate; Up-Regulation; Up-Regulation (Physiology); Upregulation; biological signal transduction; cancer cell; cancer progression; cell biology; cell motility; coated pit; disabled; disabled people; disease phenotype; epithelial carcinoma; extracellular; gene product; hypercholesterolemia; in vivo; interest; intracellular skeleton; malignancy; mammary tumor; migration; neoplasm progression; neoplasm/cancer; neoplastic progression; new technology; novel; public health medicine (field); receptor; resistant; social role; sorting; trafficking; tumor progression; tumor suppressor; uptake
Project start date: 2003-06-01
Project end date: 2012-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-07-070
5R01GM066257-07 (2010): $414691
5R01GM066257-06 (2009): $357982
SRC FAMILY KINASES AND CELL GROWTH REGULATION
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center, Box 19024, 1100 Fairview Ave N, Seattle, Wa 98109-1024
Grant 5R01CA041072-25 from National Cancer Institute
Keywords: ASV; ASVSRC1; ATP[{..}]protein-tyrosine O-phosphotransferase; Adaptor Protein; Adaptor Signaling Protein; Alleles; Allelomorphs; Animals; Antimorphic mutation; Apoptosis; Apoptosis Pathway; Binding; Binding (Molecular Function); Binding Proteins; Biochemical; Biological Function; Biological Process; Biology; Brain; C3g; CIS Family Protein; Cancers; Carcinoma; Cell Communication and Signaling; Cell Death, Programmed; Cell Locomotion; Cell Migration; Cell Movement; Cell Signaling; Cells; Cellular Migration; Cellular Regulation; Central Nervous System; Complex; Cytokine-Induced STAT Inhibitor Family Protein; Development; Dominant Negative; Dominant-Negative Mutant; Dominant-Negative Mutation; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Embryo; Embryonic; Encephalon; Encephalons; Endocytosis; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Epithelial Neoplasms, Malignant; Epithelial Tumors, Malignant; Event; Extracellular Protein; Feedback; Funding; GRF2; GTP Phosphohydrolases; GTPases; GeneHomolog; Genes, c-src; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Goals; Guanosine Triphosphate Phosphohydrolases; Guanosinetriphosphatases; HEK3; Hereditary; Homolog; Homologous Gene; Homologue; Human; Human, General; Inherited; Intracellular Communication and Signaling; Knock-in; Knock-in Mouse; Knockout Mice; Ligand Binding Protein; Link; Malignant Neoplasms; Malignant Tumor; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Medulla Spinalis; Mice; Mice, Knock-out; Mice, Knockout; Molecular Interaction; Motility; Motility, Cellular; Movement; Murine; Mus; Mutation; Nerve Cells; Nerve Unit; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neuraxis; Neurocyte; Neurons; Null Mouse; Oncogene Products; Oncogene Proteins; Oncoproteins; Outcome; PTK; Pathway interactions; Phosphorylation Site; Position; Positioning Attribute; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Protein-Tyrosine Kinases, src; Proteins; RAPGEF1 gene; RGE1; Receptor Protein; Regulation; Relative; Relative (related person); Role; SOCS Family Protein; SRC; SRC gene; SRC1; SSI Family Protein; STAT-Induced STAT Inhibitor Family Protein; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Site; Spinal Cord; Suppressor of Cytokine Signaling Family Protein; Testing; Tyrosine Kinase; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosylprotein Kinase; biological signal transduction; body movement; c src; c-src Proto-Oncogenes; cell growth regulation; cell motility; cell type; computerized data processing; data processing; epithelial carcinoma; experiment; experimental research; experimental study; gene product; genome mutation; guanosinetriphosphatase; hydroxyaryl protein kinase; in vivo; malignancy; migration; mutant; neoplasm/cancer; neuronal; novel; pathway; precursor cell; receptor; reelin receptor; research study; response; role model; shRNA; short hairpin RNA; signal processing; small hairpin RNA; social role; src Kinases; src Oncogenes; src Tyrosine Kinases; src-Family Kinases; src-Family Tyrosine Kinases; tyrosyl protein kinase; ubiquitin ligase; v src; v-SRC Avian Sarcoma (Schmidt-Ruppin A-2) Viral Oncogene Homolog; v-src Genes; v-src Oncogenes
Project start date: 1986-01-01
Project end date: 2011-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
5R01CA041072-25 (2010): $567636
Sponsored Links Excellgen http://Excellgen.com
5R37CA041072-21 (2006): $527770
5R37CA041072-20 (2005): $524731
4R37CA041072-19 (2004): $509448
5R01CA041072-11 (1996): $270569
SIGNAL TRANSDUCTION VIA RECEPTOR TYROSINE KINASES
Jonathan A Cooper, Member
Fred Hutchinson Cancer Research Center
box 19024, 1100 Fairview Ave N
seattle, Wa 981091024
Grant 5R01CA054786-06 from National Cancer Institute IRG: CBY
Project start date: 1995-09-26
Project end date: 2000-06-30
5R01CA054786-06 (1996): $231157